Applied and Environmental Microbiology最新文献

To investigate how the thickness and extracellular electron transfer (EET) capabilities of microbial biofilms influence the reduction of ferric iron [Fe(III)]-containing minerals, we utilized four strains of Geobacter sulfurreducens with varying biofilm thicknesses and EET capabilities. These strains were engineered by modulating intracellular levels of dinucleotide second messengers. We systematically investigated the capacity of biofilms formed by four strains to reduce different Fe(III)-containing minerals including ferrihydrite, goethite, and lepidocrocite. By growing the G. sulfurreducens biofilm on the Fe(III) mineral-coated slides, our results showed that the strains forming thin biofilms on surfaces of Fe(III) minerals exhibited faster Fe(III) reduction rates compared to those with thick biofilms. Transcriptomic analyses revealed the upregulation of the genes encoding bacterial EET-involved proteins in the thin biofilms, highlighting the significant role of these proteins in reducing Fe(III)-containing minerals by G. sulfurreducens biofilms. Furthermore, genetic characterization identified the participation of two novel c-type cytochromes (c-Cyts), GSU1996 and GSU2513, in the reduction of Fe(III)-containing minerals by G. sulfurreducens biofilms. The results from this study provide an improved understanding of mineral-microbe interaction.IMPORTANCEGeobacter is a predominant species within biofilm communities that facilitate iron reduction, a process essential for the biogeochemical cycling of iron and other elements. However, the specific properties of Geobacter biofilms crucial for iron reduction remain unclear. By manipulating intracellular levels of dinucleotide second messengers to generate strains with varying biofilm properties, this research reveals that thinner biofilms exhibit superior rates of ferric iron [Fe(III)] mineral reduction compared to thicker biofilms. This finding highlights the vital role of proteins involved in extracellular electron transfer (EET) in enhancing the reduction of Fe(III)-containing minerals. The study further identifies two novel c-type cytochromes, GSU1996 and GSU2513, as important contributors to this process. These discoveries not only advance our understanding of microbial iron reduction but also offer new perspectives on the interactions between biofilms and mineral surfaces, potentially informing future research and applications in biogeochemical cycling and bioenergy.

Desulfofundulus kuznetsovii strain 17^T oxidizes methanol via a two-pathway system involving both alcohol dehydrogenases (ADH) and a cobalt-dependent methanol methyltransferase (MT). In contrast, D. kuznetsovii strain TPOSR lacks the MT pathway, relying solely on ADH for growth on methanol. Despite the absence of the MT pathway, cobalt starvation resulted in lower methanol uptake rates and reduced growth rates in strain TPOSR, suggesting a critical role of cobalt in methanol metabolism outside of its role in the MT system. Given the often-crucial role of metal cofactors such as iron, zinc, and other metals in the active site of ADHs, we hypothesized that cobalt could influence the catalytic activity of the TPOSR ADHs. The gene encoding for the most abundant ADH during growth on methanol, Adh1, was heterologously expressed in Escherichia coli, and the enzyme was purified for kinetic studies. Adh1 exhibited optimal activity at 55°C and is oxygen tolerant. The methanol turnover rate increased from 1.76 (95% Cl [1.56, 1.99]) s⁻¹ to 3.5 (95% Cl [3.3, 3.72]) s⁻¹ with the addition of 2 µM CoSO₄, while higher cobalt concentrations (>5 µM) inhibited Adh1 activity. Similarly, NiSO₄ addition (1-1000 µM) enhanced Adh1 activity, with a 75% improvement observed at an optimum concentration of 200 µM. Our findings suggest that the importance of cobalt for the methanol metabolism of sulfate-reducing organisms extends beyond its involvement in the MT system.IMPORTANCEMethanol is a ubiquitous compound in natural environments, where it is produced geothermally or from plant and microbial biomass. Its microbial metabolism is particularly important in low-nutrient, oxygen-free environments, such as the deep subsurface, where specialized microbes compete for methanol and play a crucial role in the global carbon cycle. Typically, microbes in these settings rely on a cobalt-dependent methanol methyltransferase (MT) pathway for methanol breakdown. However, Desulfofundulus kuznetsovii TPOSR deviates from this, lacking the MT pathway and instead relying solely on alcohol dehydrogenases (ADH) to oxidize methanol. Despite the absence of the cobalt-dependent MT system, our study shows that cobalt strongly stimulates the activity of the most abundant ADH, revealing an unexpected, yet significant role for cobalt in this alternative methanol metabolism. Understanding these interactions not only sheds new light on methanol metabolism in nature but also opens up possibilities for developing more efficient and sustainable technologies for methanol conversion in industry.

Enterococci are lactic acid bacteria (LAB) that, as their name implies, often are found in the gastrointestinal tract of animals. Like many other gut-dwelling LAB, for example, various lactobacilli, they are frequently found in other niches as well, including plants and fermented foods from all over the world. In fermented foods, they contribute to flavor and other organoleptic properties, help extend shelf life, and some even possess probiotic properties. There are many positive attributes of enterococci; however, they have been overshadowed by the occurrence of antibiotic-resistant and virulent strains, often reported for the two species, Enterococcus faecalis and Enterococcus faecium. More than 40,000 whole-genome sequences covering 64 Enterococcus type species are currently available in the National Center for Biotechnology Information repository. Closer inspection of these sequences revealed that most represent the two gut-dwelling species E. faecalis and E. faecium. The remaining 62 species, many of which have been isolated from plants, are thus quite underrepresented. Of the latter species, we found that most carried no potential virulence and antibiotic resistance genes, an observation that is aligned with these species predominately occupying other niches. Thus, the culprits found in the Enterococcus genus mainly belong to E. faecalis, and a biased characterization has resulted in the opinion that enterococci do not belong in food. Since enterococci possess many industrially desirable traits and frequently are found in other niches besides the gut of animals, we suggest that their use as food fermentation microorganisms is reconsidered.IMPORTANCEWe have retrieved a large number of Enterococcus genome sequences from the National Center for Biotechnology Information repository and have scrutinized these for the presence of virulence and antibiotic resistance genes. Our results show that such genes are prevalently found in the two species Enterococcus faecalis and Enterococcus faecium. Most other species do not harbor any virulence and antibiotic resistance genes and display great potential for use as food fermentation microorganisms or as probiotics. The study contributes to the current debate on enterococci and goes against the mainstream perception of enterococci as potentially dangerous microorganisms that should not be associated with food and health.

Xanthomonas campestris pv. campestris (Xcc) and X. oryzae pv. oryzae (Xoo) are crucial plant pathogenic bacteria, causing crucifer black rot and rice leaf blight, respectively. Both bacterial species encode a protein containing the YiiD_C domain, designated MadB, which exhibits an 87.5% sequence identity between their MadBs. The madB genes from either Xoo or Xcc successfully restored the growth defect in Ralstonia solanacearum and Escherichia coli fabH mutants in vivo. In vitro assays demonstrated that MadB proteins possess malonyl-ACP decarboxylase activity, although Xcc MadB exhibited lower activity compared with Xoo MadB. Mutation of madB in both Xoo and Xcc strains led to decreased pathogenicity in their respective host plants. Interestingly, the Xoo madB mutant exhibited a significant increase in branched-chain fatty acid production, whereas the Xcc madB mutant showed only minor changes in fatty acid composition. Despite the reduction in exopolysaccharide (EPS) synthesis due to madB mutation in both Xoo and Xcc, EPS production in the Xoo madB mutant could be restored by exogenous sodium acetate supplementation. In contrast, sodium acetate failed to restore EPS synthesis in the Xcc madB mutant. Biochemical and genetic analyses indicated that these divergent physiological roles arise from the distinct biochemical functions of MadB in the two bacteria. In Xoo, the fatty acid synthesis (FAS) pathway mediated by MadB operates independently of the FAS pathway mediated by FabH. Conversely, in Xcc, the FAS pathway mediated by FabH is the primary route, with MadB's pathway serving a supplementary and regulatory role. Further analysis of gene organization and expression regulation of madB in both bacteria corroborates these distinctions.

Importance: Despite the high conservation of the mad gene within the Proteobacteria, the physiological roles of the Mad protein remain largely unclear. Xoo and Xcc are bacteria with very close phylogenetic relationships, both encoding malonyl-ACP decarboxylase (MadB). However, MadB demonstrates substantial physiological function variations between these two species. This study demonstrates that even in closely related bacteria, homologous genes have adopted different evolutionary pathways to adapt to diverse living environments, forming unique gene expression regulation mechanisms. This has led to the biochemical functional divergence of homologous proteins within their respective species, ultimately resulting in distinct physiological functions.

Fungi produce a wide variety of compounds, especially those that exhibit biological activity. Such compounds may aid the survival of fungi in the environment or may contribute to host infection for pathogenic species. Regarding dermatophytes, which affect a large number of patients worldwide, studies on metabolites that exhibit biological activity are scarce. In this study, to gain insight into the interaction with skin microbiota at the site of infection, we searched for compounds that exhibit antibacterial activity among the metabolites of Trichophyton rubrum. We rediscovered viomellein, a red pigment, as a potent antibacterial compound and identified its biosynthetic gene (vio) cluster by RNA-sequencing and gene deletion analyses. Sequential reconstruction of the vio cluster genes in Aspergillus oryzae revealed the biosynthetic pathway for viomellein via nor-toralactone, semivioxanthin, and vioxanthin production. The vio gene cluster is widely conserved among dermatophytes and is also present in some Aspergillus and Penicillium species. Consistent with the results, viomellein and its structural analogs, xanthomegnin, and vioxanthin, were shown to be produced by most dermatophyte species. These results suggest that dermatophytes can produce diverse naphthopyranone compounds, some of which have strong growth inhibitory effects against bacteria. This study provides a previously unknown molecular entity for antibiotic production by dermatophytes and provides insight into the interaction between commensal bacteria and dermatophytes.IMPORTANCEDermatophytes are widespread human pathogens in the world, but the mechanisms of infection have been little studied. Although bacterial density at the site of infection is abundant, interaction between dermatophytes and the bacterial community has not yet been studied. Here, to understand the infection ecology of dermatophytes, we searched for antimicrobial substances that would be effective against the dermal bacterial community. We discovered viomellein, which exhibits strong antibacterial activity against gram-positive bacteria such as Staphylococcus aureus, and its biosynthetic genes are shared not only by dermatophytes but also by other fungi. Since many dermatophytes showed the ability to produce viomellein, it is likely that this is the initial infection strategy of dermatophytes, which has been a mystery for long.

Relative humidity (RH) varies widely in indoor environments based on temperature, outdoor humidity, heating systems, and other environmental conditions. This study explored how RH affects aerosolized porcine respiratory coronavirus (PRCV), a model for coronaviruses, over a time range from 0 min to a maximum of 1 h, and the molecular mechanism behind viral infectivity reduction. These questions were answered by quantifying: (i) viral-host receptor interactions, (ii) capsid integrity, (iii) viral genome integrity, and (iv) virus infectivity. We found RH did not alter PRCV-receptor interactions. RHs 45-55% and 65-75% damaged viral genomes (2 log₁₀ reduction and 1 log₁₀ reduction, respectively, in terms of median sample value), whereas RHs 55-65% decreased capsid integrity (2 log₁₀ reduction). No apparent virion damage was observed in RH 75-85%. Two assays were used to quantify virus presence: qPCR for detecting the viral genomes and plaque-forming unit assay for detecting the virus replication. Our results indicated that the qPCR assay overestimated the concentrations of infectious viruses, and RNase treatment with long-range RT-qPCR performed better than one-step RT-qPCR. We propose that understanding the influence of RH on the stability of aerosolized viruses provides critical information for detecting and preventing the indoor transmission of coronaviruses.

Importance: Indoor environments can impact the stability of respiratory viruses, which can then affect the transmission rates. The mechanisms of how relative humidity (RH) affects virus infectivity still remain unclear. This study found RH inactivates porcine respiratory coronavirus by damaging its capsid and genome. The finding highlights the potential role of controlling indoor RH levels as a strategy to reduce the risk of coronavirus transmission.

Alphabaculoviruses produce a large number of occlusion bodies (OBs) in host cells during the late stage of infection. OBs are mainly composed of the viral product polyhedrin (POLH), and the extremely high-level transcription of the polh gene has been exploited to express foreign proteins in insect cultured cells, larvae, and pupae. This polh hyper-transcription requires the "burst sequence" located between the transcriptional start site and the initiation codon. Here, we focused on the roles of the A-rich region within the burst sequence. We generated Bombyx mori nucleopolyhedrovirus mutants whose burst sequence contained "A-to-T" mutations in the A-rich region. Some mutants exhibited levels of polh promoter-driven reporter expression lower than or comparable to that of the wild type, whereas the mutants with TTT mutations at positions -16 to -14 in the polh upstream region showed a four- to fivefold increase in it. Most cases of single or double A-to-T mutations at -16 to -14 of the upstream region had small but significant effects on the expression level, while the triple mutation was the most effective. This enhancement was also observed in the Autographa californica multiple nucleopolyhedrovirus-based vector system, which is more commonly used for foreign protein expression. We also found that this triple mutation enhanced the accumulation of polh mRNA and POLH protein even in an OB-producing virus. These results indicate that specific mutations in the burst sequence have the potential to increase baculoviral protein expression at the transcriptional level and may improve foreign protein expression by baculoviruses.IMPORTANCEThe most notable characteristic of alphabaculoviruses is that they produce many proteinaceous occlusion bodies (OBs) during the very late stages of infection. The main component of these OBs is virus-encoded polyhedrin (POLH). The high expression of the polh gene led to the development of the baculovirus expression vector system (BEVS). Currently, this system is widely used for the production of vaccines, veterinary medicines, and reagents. Despite this background, the mechanisms by which baculoviruses ultimately produce large quantities of OBs remain largely unexplained, even after approximately 40 years since the BEVS development. Here, we discovered that three nucleotide substitutions in the polh burst sequence markedly increased the polh expression levels in both BmNPV- and AcMNPV-based BEVSs, regardless of the vector type. This discovery can be easily introduced into the currently used BEVS, possibly contributing to further improvements for achieving even higher expression of foreign proteins.

Society relies heavily on chemicals traditionally produced through the refinement of fossil fuels. The conversion of renewable biomass to value-added chemicals by microbes, particularly hyperthermophiles (T_opt ≥80°C), offers a renewable alternative to this traditional approach. Herein, we describe the engineering of the hyperthermophilic archaeon Pyrococcus furiosus, which grows optimally (T_opt) at 100°C, for the conversion of sugar to 1-propanol. This was accomplished by constructing a hybrid metabolic pathway consisting of two native and seven heterologously produced enzymes to convert acetyl-CoA from carbohydrate metabolism to 1-propanol. A total of eleven foreign genes from two other organisms were utilized, one from the thermophilic bacterium Thermoanaerobacter sp. strain X514 and 10 from the thermoacidophilic archaeon Metallosphaera sedula, both of which grow optimally near 70°C. The recombinant P. furiosus strain produced 1-propanol at similar concentrations (up to ~1 mM) when incubated at 75°C to activate the gene products of Thermoanaerobacter sp. strain X514 and M. sedula and by initially incubating at 95°C for P. furiosus growth and then subsequently returning to 75°C to promote 1-propanol formation. Note that 1-propanol was not produced if the culture was grown only at 95°C. This work has the potential for future optimization through harnessing the genome-scale metabolic model of P. furiosus that was used herein to identify engineering targets to increase 1-propanol titer.IMPORTANCEAs petroleum reserves become increasingly strained, the development of renewable alternatives to traditional chemical synthesis becomes more important. In this work, a high-temperature biological system for sugar to 1-propanol conversion was demonstrated by metabolic engineering of the hyperthermophilic archaeon Pyrococcus furiosus (T_opt 100°C). The engineered strain produced 1-propanol by temperature shifting from 75°C to 95°C and then back to 75°C to accommodate the temperature ranges for native and foreign proteins associated with 1-propanol biosynthesis. Genome-scale metabolic modeling informed the carbon and reductant flux in the system, identified potential factors limiting 1-propanol production, and revealed potential optimization targets.